Ultrasonic imaging system utilizing a long-persistence contrast agent

ABSTRACT

A microbubble preparation formed of a plurality of microbubbles comprising a first gas and second gas surrounded by a membrane such as a surfactant, wherein the first gas and the second gas are present in a molar ration of from about 1:100 to abut 1000:1, and wherein the first gas has a vapor pressure of at least about (760−X) mm Hg at 37° C., where x is the vapor pressure of the second gas at 37° C., and wherein the vapor pressure of each of the first and second gases is greater than about 75 mm Hg at 37° C.; also disclosed are methods for preparing microbubble compositions, including compositions that rapidly shrink from a first average diameter to a second average diameter less than about 75% of the first average diameter and are stabilized at the second average diameter; methods and kits for preparing microbubbles; and methods for using such microbubbles as contrast agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/919,433 filedJul. 30, 2001 now abandoned, which is a continuation of U.S. Ser. No.08/486,531 filed Jun. 7, 1995 now U.S Pat. No. 5,689,741, which is adivisional of U.S. Ser. No. 08/284,083, filed Aug. 1, 1994 now U.S. Pat.No. 5,605,673, which is a continuation-in-part of U.S. application Ser.No. 08/099,951, filed Jul. 30, 1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention includes a method for preparing stable long-livedmicrobubbles for ultrasound contrast enhancement and other uses, and tocompositions of the bubbles so prepared.

2. Background of the Art

Ultrasound technology provides an important and more economicalalternative to imaging techniques which use ionizing radiation. Whilenumerous conventional imaging technologies are available, e.g., magneticresonance imaging (MRI), computerized tomography (CT), and positronemission tomography (PET), each of these techniques use extremelyexpensive equipment. Moreover, CT and PET utilize ionizing radiation.Unlike these techniques, ultrasound imaging equipment is relativelyinexpensive. Moreover, ultrasound imaging does not use ionizingradiation.

Ultrasound imaging makes use of differences in tissue density andcomposition that affect the reflection of sound waves by those tissues.Images are especially sharp where there are distinct variations intissue density or compressibility, such as at tissue interfaces.Interfaces between solid tissues, the skeletal system, and variousorgans and/or tumors are readily imaged with ultrasound.

Accordingly, in many imaging applications ultrasound performs suitablywithout use of contrast enhancement agents; however, for otherapplications, such as visualization of flowing blood, there have beenongoing efforts to develop such agents to provide contrast enhancement.One particularly significant application for such contrast agents is inthe area of perfusion imaging. Such ultrasound contrast agents couldimprove imaging of flowing blood in the heart muscle, kidneys, lungs,and other tissues. This, in turn, would facilitate research, diagnosis,surgery, and therapy related to the imaged tissues. A blood poolcontrast agent would also allow imaging on the basis of blood content(e.g., tumors and inflamed tissues) and would aid in the visualizationof the placenta and fetus by enhancing only the maternal circulation.

A variety of ultrasound contrast enhancement agents have been proposed.The most successful agents have generally consisted of microbubbles thatcan be injected intravenously. In their simplest embodiment,microbubbles are miniature bubbles containing a gas, such as air, andare formed through the use of foaming agents, surfactants, orencapsulating agents. The microbubbles then provide a physical object inthe flowing blood that is of a different density and a much highercompressibility than the surrounding fluid tissue and blood. As aresult, these microbubbles can easily be imaged with ultrasound.

Most microbubble compositions have failed, however, to provide contrastenhancement that lasts even a few seconds, let alone minutes, ofcontrast enhancement. This greatly limits their usefulness. Microbubbleshave therefore been “constructed” in various manners in an attempt toincrease their effective contrast enhancement life. Various avenues havebeen pursued: use of different surfactants or foaming agents; use ofgelatins or albumin microspheres that are initially formed in liquidsuspension, and which entrap gas during solidification; and liposomeformation. Each of these attempts, in theory, should act to createstronger bubble structures. However, the entrapped gases (typically air,CO₂, and the like) are under increased pressure in the bubble due to thesurface tension of the surrounding surfactant, as described by theLaPlace equation (ΔP=2γ/r).

This increased pressure, in turn, results in rapid shrinkage anddisappearance of the bubble as the gas moves from a high pressure area(in the bubble) to a lower pressure environment (in either thesurrounding liquid which is not saturated with gas at this elevatedpressure, or into a larger diameter, lower pressure bubble).

Solid phase shells that encapsulate gases have generally proven toofragile or too permeable to the gas to have satisfactory in vivo life.Furthermore, thick shells (e.g., albumin, sugar, or other viscousmaterials) reduce the compressibility of the bubbles, thereby reducingtheir echogenicity during the short time they can exist. Solid particlesor liquid emulsion droplets that evolve gas or boil when injected posethe danger of supersaturating the blood with the gas or vapor. This willlead to a small number of large embolizing bubbles forming at the fewavailable nucleation sites rather than the intended large number ofsmall bubbles.

One proposal for dealing with such problems is outlined in Quay,PCT/US92/07250. Quay forms bubbles using gases selected on the basis ofbeing a gas at body temperature (below 37° C.), and having reduced watersolubility, higher density, and reduced gas diffusivity in solution incomparison to air. Although reduced water solubility and diffusivity canaffect the rate at which the gas leaves the bubble, numerous problemsremain with the Quay bubbles. Forming bubbles of sufficiently smalldiameter (e.g., 3-5 μm) requires high energy input. This is adisadvantage in that sophisticated bubble preparation systems must beprovided at the site of use. Moreover, The Quay gas selection criteriaare incorrect in that they fail to consider certain major causes ofbubble shrinkage, namely, the effects of bubble surface tension,surfactants and gas osmotic effects, and these errors result in theinclusion of certain unsuitable gases and the exclusion of certainoptimally suitable gases.

Accordingly, a need exists in the art for compositions, and a method toprepare such compositions, that provide, or utilize, a longer lifecontrast enhancement agent that is biocompatible, easily prepared, andprovides superior contrast enhancement in ultrasound imaging.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided anultrasound contrast enhancement agent that has a prolonged longevity invivo, which consists of virtually any conventional microbubbleformulation in conjunction with an entrapped gas or gas mixture that isselected based upon consideration of partial pressures of gases bothinside and outside of the bubble, and on the resulting differences ingas osmotic pressure that oppose bubble shrinkage. Gases having a lowvapor pressure and limited solubility in blood or serum (i.e.,relatively hydrophobic) may advantageously be provided in combinationwith another gas that is more rapidly exchanged with gases present innormal blood or serum. Surfactant families allowing the use of highermolecular weight gas osmotic agents, and improved methods of bubbleproduction are also disclosed.

One aspect of the present invention is a stabilized gas filledmicrobubble preparation, comprising a mixture of a first gas or gasesand a second gas or gases (gas osmotic agent) within generally sphericalmembranes to form microbubbles, wherein the first gas and the second gasare respectively present in a molar ratio of about 1:100 to about1000:1, and wherein the first gas has a vapor pressure of at least about(760−x) mm Hg at 37° C., where x is the vapor pressure of the second gasat 37° C., and wherein the vapor pressure of each of the first andsecond gases is greater than about 75 mm Hg at 37° C., with the provisothat the first gas and the second gas are not water vapor. In oneembodiment, the second gas comprises a fluorocarbon and the first gas isa nonfluorocarbon, such as nitrogen, oxygen, carbon dioxide, or amixture thereof.

The microbubbles may advantageously be provided in a liquid medium, suchas an aqueous medium, wherein they have a first average diameter, theratio of the first gas to the second gas in the microbubbles is at least1:1, and the microbubbles are adapted to shrink in the medium as aresult of loss of the first gas through the membrane to a second averagediameter of less than about 75% of the first diameter and then remainstabilized at or about the second diameter for at least about 1 minuteas a result of a gas osmotic pressure differential across the membrane.Advantageously, the medium is in a container and the microbubbles haveactually been formed in the container. Alternatively, the medium isblood in vivo. In one embodiment, the liquid medium contains gas orgases dissolved therein with a gas tension of at least about 700 mm Hg,wherein the first diameter is at least about 5 μm, and wherein thetension of the gas or gases dissolved in the medium is less than thepartial pressure of the same gas or gases inside the microbubbles.

In a particularly preferred embodiment, the bubble initially contains atleast three gases: a first gas having a partial pressure far greaterthan the gas tension of the same gas in the surrounding liquid (e.g.,1.5, 2, 4, 5, 10, 20, 50, or 100 or more times greater than in thesurrounding liquid); a second gas that is retained in the bubble due toa relatively low permeability of the bubble membrane to the gas, or arelatively low solubility of the gas in the surrounding medium (asdescribed elsewhere herein), and a third gas to which the membrane isrelatively permeable that is also present in the surrounding medium. Forexample, in an aqueous system exposed to or at least partiallyequilibrated with air (such as blood), the first gas may advantageouslybe carbon dioxide or another gas not present in large quantities in airor blood; the second gas may be a fluorocarbon gas, such asperfluorohexane; and the third gas may be air or a major component ofair such as nitrogen or oxygen.

Preferably, the first diameter prior to shrinkage is at least about 10μm and the second diameter at which the diameter is stabilized isbetween about 1 μm and 6 μm.

For all of the microbubble preparations or methods described herein, inone preferred embodiment, the second gas has an average molecular weightat least about 4 times that of the first gas. In another preferredembodiment, the second gas has a vapor pressure less than about 750 or760 mm Hg at 37° C. Moreover, it is preferred that the molar ratio ofthe first gas to the second gas is from about 1:10 to about 500:1,200:1, or 100:1. In other preferred embodiments, the second gascomprises a fluorocarbon or a mixture of at least two or threefluorocarbons, and the first gas is a nonfluorocarbon. In someadvantageous preparations, the second gas comprises one or moreperfluorocarbons. In others, both the first gas and the second gascomprise fluorocarbons. In still others, the microbubbles contain as thefirst gas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluorohexane in a ratio from about1:10 to about 10:1. Alternatively, the microbubbles contain as the firstgas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluoropentane in a ratio fromabout 1:10 to about 10:1. It is advantageous that the second gas leavethe microbubble much more slowly than does the first gas; thus, it ispreferred that the second gas has a water solubility of not more thanabout 0.5 mM at 25° C. and one atmosphere, and the first gas has a watersolubility at least about 10 times, and preferably at least 20, 50, 100,or 200 times greater than that of the second gas. Similarly, it ispreferred that the permeability of the membrane to the first gas is atleast about 5 times, preferably 10, 20, 50, or 100 times greater thanthe permeability of the membrane to the second gas.

The microbubble preparation may advantageously be contained in acontainer, having a liquid in the container in admixture with themicrobubbles, wherein the container further comprises means fortransmission of sufficient ultrasonic energy to the liquid to permitformation of the microbubbles by sonication. In this way, themicrobubbles can be formed by the physician (or other professional)immediately before use by applying ultrasonic energy from an outsidesource to the sterile preparation inside the container. This means fortransmission can, for example, be a flexible polymer material having athickness less than about 0.5 mm (which permits ready transmission ofultrasonic energy without overheating the membrane). Such membranes canbe prepared from such polymers as natural or synthetic rubber or otherelastomer, polytetrafluoroethylene, polyethylene terephthalate, and thelike.

In the microbubble preparations of the invention, the membrane enclosingthe gas is preferably a surfactant. One preferred type of surfactantcomprises a non-Newtonian viscoelastic surfactant, alone or incombination with another surfactant. Other preferred general andspecific categories of surfactants include carbohydrates, such aspolysaccharides, derivatized carbohydrates, such as fatty acid esters ofsugars such as sucrose (preferably sucrose stearate), and proteinaceoussurfactants including albumin. Alternatively, the membrane of themicrobubble need not be a fluid (such as a surfactant), but instead canbe a solid or semi-solid, such as hardened, thickened, or denaturedproteinaceous material (e.g. albumin), carbohydrates, and the like.

One advantageous form of the invention is a kit for use in preparingmicrobubbles, preferably at the site of use. This kit may comprisesealed container (such as a vial with a septum seal for easy removal ofthe microbubbles using a hypodermic syringe), a liquid in the container(such as water or a buffered, isotonic, sterile aqueous medium), asurfactant in the container, and a fluorocarbon gas (including afluorocarbon vapor) in the container, wherein the liquid, thesurfactant, and the fluorocarbon gas or vapor are together adapted toform microbubbles upon the application of energy thereto. The energyadvantageously may be simple shaking energy, either manual ormechanical, stirring or whipping, or ultrasonic energy. The kitpreferably includes means in the container for permitting transmissionof sufficient external ultrasonic energy to the liquid to formmicrobubbles in the container. As above, the means for transmission canin one embodiment comprise a flexible polymer membrane having athickness less than about 0.5 mm. In one embodiment, the kit furtherincludes a nonfluorocarbon gas in the container, wherein the molar ratioof the nonfluorocarbon gas to the fluorocarbon gas is from about 1:10 toabout 1000:1, with the proviso that the nonfluorocarbon gas is not watervapor. In all of the kits of the present invention, the surfactant, thegas or gases, and the other elements of the kit may in some embodimentsbe the same as recited above for the microbubble preparation per se.

In another embodiment, the kit comprises a container, driedliquid-soluble void-containing structures in the container, thevoid-containing structures defining a plurality of voids having anaverage diameter less than about 100 μm, a gas in the voids, and asurfactant, wherein the void-containing structures, the gas, and thesurfactant are together adapted to form microbubbles upon addition tothe container of a liquid in which the void-containing structures aresoluble. These void-containing structures can be made at least in partof the surfactant, e.g., by lyophilization of void-forming material orby spray drying, or can be formed from any other liquid soluble(preferably water soluble) film-forming material, such as albumin,enzymes, or other proteins, simple or complex carbohydrates orpolysaccharides, and the like. The surfactants used in the kit canadvantageously be those described above in connection with themicrobubble preparations per se.

The present invention also includes a method for forming microbubbles,comprising the steps of providing a first gas, a second gas, a membraneforming material, and a liquid, wherein the first gas and the second gasare present in a molar ratio of from about 1:100 to about 1,000:1, andwherein the first gas has a vapor pressure of at least about (760−x) mmHg at 37° C., where x is the vapor pressure of the second gas at 37° C.,and wherein the vapor pressure of each of the first and second gases isgreater than about 75 mm Hg at 37° C., with the proviso that the firstgas and the second gas are not water vapor, and surrounding the firstand second gases with the membrane forming material to form microbubblesin the liquid. The membrane forming materials and gases may be asdescribed above. The method preferably further comprises the steps ofinitially forming microbubbles having a first average diameter whereinthe initial ratio of the first gas to the second gas in the microbubblesis at least about 1:1, contacting the microbubbles having a firstaverage diameter with a liquid medium, shrinking the microbubbles in themedium as a result of loss of the first gas through the membrane, andthen stabilizing the microbubbles at a second average diameter of lessthan about 75% of the first diameter for a period of at least oneminute. Preferably, the microbubbles are stabilized at the seconddiameter by providing a gas osmotic pressure differential across themembrane such that the tension of a gas or gases dissolved in the mediumis greater than or equal to the pressure of the same gas or gases insidethe microbubbles. In one embodiment, the first diameter is at leastabout 5 μm.

The invention also includes a method for forming microbubbles,comprising the steps of providing dried liquid-soluble void-containingstructures, the void-containing structures defining a plurality of voidshaving a diameter less than about 100 μm, providing a gas in the voids,providing a surfactant, combining together the void-containingstructures, the gas, the surfactant, and a liquid in which thevoid-containing structures are soluble, and dissolving thevoid-containing structures in the liquid whereby the gas in theenclosures forms microbubbles that are surrounded by the surfactant. Aswith the kit, preferred void-containing structures are formed ofprotein, surfactant, carbohydrate, or any of the other materialsdescribed above.

Another aspect of the present invention is a method for producingmicrobubbles having increased in-vial stability, comprising the steps ofspray drying a liquid formulation of a biocompatible material to producehollow microspheres therefrom, permeating the microspheres with a gasosmotic agent (the second gas) mixed with the first gas and storing themicrospheres in a container with the gas mixture, and then adding anaqueous phase to the powder, wherein the powder dissolves in the aqueousphase to entrap the gas mixture within a liquid surfactant membrane toform microbubbles. In one embodiment, the microsphere comprises a starchor starch derivative having a molecular weight of greater than about500,000 or a dextrose equivalency value less than about 12. Onepreferred starch derivative is hydroxyethyl starch. Preferably, the gasosmotic agent comprises a perfluorocarbon. In another embodiment, themicrosphere comprises a sugar ester having a component with ahydrophilic-lipophilic balance less than about eight. Preferably, thesugar ester is sucrose tristearate.

The present invention also includes a method for forming microbubbles,comprising the step of providing a gas osmotic agent-permeatedsurfactant powder, and combining the powder with an aqueous phase.

Still another aspect of the present invention is a method for increasingthe in vivo half-life of microbubbles, comprising providing a spraydried formulation in combination with a gas osmotic agent that permeatesthe formulation, and combining the formulation with an aqueous phase toform microbubbles having a half-life in vivo of at least about 20seconds. In preferred embodiments, the spray dried formulation comprisesa starch or starch derivative and the sugar ester is sucrosetristearate.

Finally, the present invention includes a method for imaging an objector body, comprising the steps of introducing into the object or body anyof the aforementioned microbubble preparations and then imaging at leasta portion of the object or body via ultrasound or magnetic resonance.Preferably, the body is a vertebrate and the preparation is introducedinto the vasculature of the vertebrate. The method may further includepreparing the microbubbles in any of the aforementioned manners prior tointroduction into the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a two-chamber vial containing amicrobubble-forming preparation, with an aqueous solution in an upperchamber and solid and gaseous ingredients in a lower chamber.

FIG. 2 illustrates the vial of FIG. 1, where the aqueous solution hasbeen mixed with the solid ingredients to form microbubbles foradministration to a patient.

FIG. 3 is a perspective view of an inverted two-chamber vial containinga microbubble-forming preparation, with an aqueous solution in the lowerchamber and solid and gaseous ingredients in the upper chamber.

FIG. 4 illustrates the vial of FIG. 3, where the aqueous solution hasbeen mixed with the solid ingredients to form microbubbles foradministration to a patient.

DETAILED DESCRIPTION OF THE INVENTION

As used in the present description and claims, the terms “vapor” and“gas” are used interchangeably. Similarly, when referring to the tensionof dissolved gas in a liquid, the more familiar term “pressure” may beused interchangeably with “tension.” “Gas osmotic pressure” is morefully defined below, but in a simple approximation can be thought of asthe difference between the partial pressure of a gas inside amicrobubble and the pressure or tension of that gas (either in a gasphase or dissolved in a liquid phase) outside of the microbubble, whenthe microbubble membrane is permeable to the gas. More precisely, itrelates to differences in gas diffusion rates across a membrane. Theterm “membrane” is used to refer to the material surrounding or defininga microbubble, whether it be a surfactant, another film forming liquid,or a film forming solid or semisolid. “Microbubbles” are considered tobe bubbles having a diameter between about 0.5 and 300 μm, preferablyhaving a diameter no more than about 200, 100, or 50 μm, and forintravascular use, preferably not more than about 10, 8, 7, 6, or 5 μm(measured as average number weighted diameter of the microbubblecomposition). When referring to a “gas,” it will be understood thatmixtures of gases together having the requisite property fall within thedefinition, except where the context otherwise requires. Thus, air maytypically be considered a “gas” herein.

The present invention provides microbubbles that have a prolongedlongevity in vivo that are suitable for use as ultrasound and magneticresonanace imaging (MRI) contrast enhancement agents. Typical ultrasoundcontrast enhancement agents exhibit contrast enhancement potential foronly about one pass through the arterial system, or a few seconds toabout a minute, and thus do not survive past the aorta in a patientfollowing intravenous injection. In comparison, contrast agents preparedin accordance with the present invention continue to demonstratecontrast enhancements lives measured in multiple passes through theentire circulatory system of a patient following intravenous injection.Bubble lives of several minutes are easily demonstrated. Suchlengthening of contrast enhancement potential during ultrasound ishighly advantageous. In addition, the contrast enhancement agents of theinvention provide superior imaging; for example, clear, vivid, anddistinct images of blood flowing through the heart, lungs, and kidneysare achieved. Thus small, nontoxic doses can be administered in aperipheral vein and used to enhance images of the entire body.

As disclosed in U.S. Pat. No. 5,315,997, gases and perfluorocarbonvapors have magnetic susceptibilities substantially different fromtissues and blood. Therefore, the microbubbles of the present inventionwill cause changes in the local magnetic fields present in tissues andblood during MRI. These changes can be discerned on an MRI image and canbe used to detect the presence of a contrast agent. The agent of theinvention will persist in the blood pool longer and will therefore allowlonger, more sensitive scans of larger portions of the body.

While bubbles have been shown to be the most efficient ultrasoundscatterers for use in intravenous ultrasound contrast agents, their mainpractical drawback is the extremely short lifetime of the small(typically less than 5 microns diameter) bubbles required to passthrough capillaries in suspension. This short lifetime is caused by theincreased gas pressure inside the bubble, which results from the surfacetension forces acting on the bubble. This elevated internal pressureincreases as the diameter of the bubble is reduced. The increasedinternal gas pressure forces the gas inside the bubble to dissolve,resulting in bubble collapse as the gas is forced into solution. TheLaPlace equation, ΔP=2γ/r, (where ΔP is the increased gas pressureinside the bubble, γ is the surface tension of the bubble film, and r isthe radius of the bubble) describes the pressure exerted on a gas bubbleby the surrounding bubble surface or film. The LaPlace pressure isinversely proportional to the bubble radius; thus, as the bubbleshrinks, the LaPlace pressure increases, increasing the rate ofdiffusion of gas out of the bubble and the rate of bubble shrinkage.

It was surprisingly discovered that gases and gas vapor mixtures whichcan exert a gas osmotic pressure opposing the LaPlace pressure cangreatly retard the collapse of these small diameter bubbles. In general,the invention uses a primary modifier gas or mixture of gases thatdilute a gas osmotic agent to a partial pressure less than the gasosmotic agent's vapor pressure until the modifier gas will exchange withgases normally present in the external medium. The gas osmotic agent oragents are generally relatively hydrophobic and relatively bubblemembrane impermeable and also further possess the ability to develop gasosmotic pressures greater than 75 or 100 Torr at a relatively low vaporpressure.

The process of the invention is related to the well known osmotic effectobserved in a dialysis bag containing a solute that is substantiallymembrane impermeable (e.g. PEG, albumin, polysaccharide, starch)dissolved in an aqueous solution is exposed to a pure water externalphase. The solute inside the bag dilutes the water inside the bag andthus reduces the rate of water diffusion out of the bag relative to therate of pure water (full concentration) diffusion into the bag. The bagwill expand in volume until an equilibrium is established with anelevated internal pressure within the bag which increases the outwarddiffusional flux rate of water to balance the inward flux rate of thepure water. This pressure difference is the osmotic pressure between thesolutions.

In the above system, the internal pressure will slowly drop as thesolute slowly diffuses out of the bag, thus reducing the internal soluteconcentration. Other materials dissolved in the solution surrounding thebag will reduce this pressure further, and, if they are more effectiveor at a higher concentration, will shrink the bag.

It was observed that bubbles of air saturated with selectedperfluorocarbons grow rather than shrink when exposed to air dissolvedin a liquid due to the gas osmotic pressure exerted by theperfluorocarbon vapor. The perfluorocarbon vapor is relativelyimpermeable to the bubble film and thus remains inside the bubble. Theair inside the bubble is diluted by the perfluorocarbon, which acts toslow the air diffusion flux out of the bubble. This gas osmotic pressureis proportional to the concentration gradient of the perfluorocarbonvapor across the bubble film, the concentration of air surrounding thebubble, and the ratio of the bubble film permeability to air and toperfluorocarbon.

As discussed above, the LaPlace pressure is inversely proportional tothe bubble radius; thus, as the bubble shrinks, the LaPlace pressureincreases, increasing the rate of diffusion of gas out of the bubble andthe rate of bubble shrinkage, and in some cases leading to thecondensation and virtual disappearance of a gas in the bubble as thecombined LaPlace and external pressures concentrate the osmotic agentuntil its partial pressure reaches the vapor pressure of liquid osmoticagent.

We have discovered that conventional microbubbles that contain anysingle gas will subsist in the blood for a length of time that dependsprimarily on the arterial pressure, the bubble diameter, the membranepermeability of the gas through the bubble's surface, the mechanicalstrength of the bubble's surface, the presence, absence, andconcentration of the gases that are ordinarily present in the blood orserum, and the surface tension present at the surface of the bubble(which is primarily dependent on the diameter of the bubble andsecondarily dependent on the identity and concentration of thesurfactants which form the bubble's surface). Each of these parametersare interrelated, and they interact in the bubble to determine thelength of time that the bubble will last in the blood.

The present invention includes the discovery that a single gas or acombination of gases can together act to stabilize the structure of themicrobubbles entraining or entrapping them. Essentially, the inventionutilizes a first gas or gases (a “primary modifier gas”) that optionallyis ordinarily present in normal blood and serum in combination with oneor more additional second gases (a “gas osmotic agent or agents” or a“secondary gas”) that act to regulate the osmotic pressure within thebubble. Through regulating the osmotic pressure of the bubble, the gasosmotic agent (defined herein as a single or mixture of chemicalentities) exerts pressure within the bubble, aiding in preventingdeflation. Optionally, the modifier gas may be a gas that is notordinarily present in blood or serum. However, the modifier gas must becapable of diluting and maintaining the gas osmotic agent or agents at apartial pressure below the vapor pressure of the gas osmotic agent oragents while the gases in blood or other surrounding liquid diffuse intothe bubble. In an aqueous medium, water vapor is not considered to beone of the “gases” in question. Similarly, when microbubbles are in anonaqueous liquid medium, the vapor of that medium is not considered tobe one of the “gases.”

We have discovered that by adding a gas osmotic agent that has, forexample, a reduced membrane permeability through the bubble's surface orreduced solubility in the external continuous phase liquid phase, thelife of a bubble formed therewith will be radically increased. Thisstabilizing influence can be understood more readily through adiscussion of certain theoretical bubbles. First, we will consider theeffects of arterial pressure and surface tension on a hypotheticalmicrobubble containing only air.

Initially, a hypothetical bubble containing only air is prepared. Forpurposes of discussion, this bubble will initially be considered to haveno LaPlace pressure. Generally, when equilibrated at standardtemperature and pressure (STP), it will have a internal pressure of 760Torr of air and the surrounding fluid air concentration will also beequilibrated at 760 Torr (i.e., the fluid has an air tension of 760Torr). Such a bubble will neither shrink nor grow.

Next, when the above hypothetical bubble is introduced into the arterialsystem, the partial pressure of air (or air tension) in the blood (theair pressure at which the blood was saturated with air) will also beapproximately 760 Torr and there will be an arterial pressure (for thepurposes of this discussion at 100 Torr). This total creates an externalpressure on the bubble of 860 Torr, and causing the gases in the bubbleto be compressed until the internal pressure increases to 860 Torr.There then arises a difference of 100 Torr between the air pressureinside the bubble and the air tension of the fluid surrounding thebubble. This pressure differential causes air to diffuse out of thebubble, through its air-permeable surface membrane, causing the bubbleto shrink (i.e., lose air) as it strives to reach equilibrium. Thebubble shrinks until it disappears.

Next, consider the additional, and more realistic, effect on thehypothetical bubble of adding the surface tension of the bubble. Thesurface tension of the bubble leads to a LaPlace pressure exerted on gasinside the bubble. The total pressure exerted on the gas inside thebubble is computed through adding the sum of the atmospheric pressure,the arterial pressure and the LaPlace pressure. In a 3 μm bubble asurface tension of 10 dynes per centimeter is attainable with wellchosen surfactants. Thus, the LaPlace pressure exerted on thehypothetical 3 μm bubble is approximately 100 Torr and, in addition, thearterial pressure of 100 Torr is also exerted on the bubble. Therefore,in our hypothetical bubble, the total external pressure applied to thegas inside the bubble is 960 Torr.

The bubble will be compressed until the pressure of the air inside thebubble rises to 960 Torr. Accordingly, a concentration differential of200 Torr arises between the air inside the bubble and the air dissolvedin the blood. Therefore, the bubble will rapidly shrink and disappeareven more rapidly than it did in the previous case, as it attempts toreach equilibrium.

The discovery of the present invention is illustrated by considering athird hypothetical microbubble containing air and a gas osmotic agent ora secondary gas. Assume that a theoretical bubble, initially having noarterial pressure and no LaPlace pressure, is prepared having a totalpressure of 760 Torr, which is made up of air at a partial pressure of684 Torr and a perfluorocarbon (“PFC”) as a gas osmotic agent at apartial pressure of 76 Torr. Further, assume that the perfluorocarbon isselected to have one or more traits that make it capable of acting as anappropriate gas osmotic agent, such as limited bubble membranepermeability or limited solubility in the external liquid phase. Thereis an initial gas osmotic pressure differential between the 684 Torr ofair within the bubble and the 760 Torr of air tension outside the bubble(assuming STP) of 76 Torr. This 76 Torr initial pressure difference isthe initial gas osmotic pressure and will cause the bubble to expand.Air from outside of the bubble will diffuse into and inflate the bubble,driven by the osmotic pressure differential, similar to the way waterdiffuses into a dialysis bag containing a starch solution, and inflatesthe bag.

The maximum gas osmotic pressure this gas mixture can develop is relatedto the partial pressure of the PFC and the ratio of the permeability ofthe PFC to the permeability of the air in the surrounding fluid. Intheory, and as observed experimentally, the bubble will growindefinitely as the system attempts to reach osmotic equilibrium betweenthe concentration of air (equivalent to the partial pressure of air)within the bubble and the concentration of air surrounding the bubble(the air tension).

When the hypothetical mixed gas bubble is exposed to 100 Torr ofarterial pressure where the blood has a dissolved air tension of 760Torr, the total external pressure will equal 860 Torr (760 Torratmospheric pressure and 100 Torr arterial pressure). The bubble willcompress under the arterial pressure, causing the internal pressure ofthe bubble to reach 860 Torr. The partial pressure of the air willincrease to 774 Torr and the partial pressure of the PFC (the secondgas) will increase to 86 Torr. The air will diffuse out of the bubbleuntil it reaches osmotic equilibrium with the air dissolved in the blood(i.e., 760 Torr) and the partial pressure of the PFC will increase to100 Torr. The partial pressure of the PFC will act to counterbalance thepressure exerted due to the arterial pressure, halting shrinkage of thebubble, in each case, assuming that the permeability of the bubble tothe PFC is negligible.

When the surface tension or LaPlace pressure component of 100 Torr isadded (as discussed above with the air bubble), a total of 200 Torradditional pressure is exerted on the gas in the bubble. Again, thebubble will compress until the pressure inside the bubble increases to960 Torr (partial pressure of air 864 and partial pressure of PFC 96).The air will diffuse from the bubble until it reaches 760 Torr (inequilibrium with the concentration of air the dissolved in the blood)and the partial pressure of the PFC will increase to 200 Torr, where,again, the gas osmotic pressure induced by the PFC will act tocounterbalance the pressure exerted by the LaPlace pressure and thearterial pressure, again, assuming that the membrane permeability of thebubble to the PFC is negligible.

Similarly, if the partial pressure of air in the bubble is lower thanthe air tension in the surrounding liquid, the bubble will actually growuntil the PFC is sufficiently diluted by incoming air so that thepressure of air inside and the air tension outside of the bubble areidentical.

Thus, bubbles can be stabilized effectively through the use ofcombinations of gases, since the correct combination of gases willresult in a gas osmotic pressure differential that can be harnessed tocounterbalance the effects of the LaPlace pressure and the arterialpressure exerted on the gas within the bubble in circulating blood.

Examples of particular uses of the microbubbles of the present inventioninclude perfusion imaging of the heart, the myocardial tissue, anddetermination of perfusion characteristics of the heart and its tissuesduring stress or exercise tests, or perfusion defects or changes due tomyocardial infarction. Similarly, myocardial tissue can be viewed afteroral or venous administration of drugs designed to increase the bloodflow to a tissue. Also, visualization of changes in myocardial tissuedue to or during various interventions, such as coronary tissue veingrafting, coronary angioplasty, or use of thrombolytic agents (TPA orstreptokinase) can also be enhanced. As these contrast agents can beadministered conveniently via a peripheral vein to enhance thevisualization of the entire circulatory system, they will also aid inthe diagnosis of general vascular pathologies and in the ability tomonitor the viability of placental tissue ultrasonically.

It should, however, be emphasized that these principles have applicationbeyond ultrasound imaging. Indeed, the present invention is sufficientlybroad to encompass the use of gas osmotic pressure to stabilize bubblesfor uses in any systems, including nonbiological applications.

In a preferred embodiment, the microbubbles of the present inventionhave a surfactant-based bubble membrane. However, the principles of theinvention can be applied to stabilize microbubbles of virtually anytype. Thus, mixed gases or vapors of the type described above canstabilize albumin based bubbles, polysaccharide based microbubbles,spray dried microsphere derived microbubbles, and the like. This resultis achieved through the entrapment, within the chosen microbubble, of acombination of gases, preferably a primary modifier gas or mixture ofgases that will dilute a gas osmotic agent to a partial pressure lessthan the gas osmotic agent's vapor pressure until the modifier gas willexchange with gases normally present in the external medium. The gasosmotic agent or agents are generally relatively hydrophobic andrelatively bubble membrane impermeable and also further possess theability to develop gas osmotic pressures greater than 50, 75, or 100Torr. In one preferred embodiment, the gas vapor pressure of the gasosmotic agent is preferably less than about 760 Torr at 37° C.,preferably less than about 750, 740, 730, 720, 710, or 700 Torr, and insome embodiments less than about 650, 600, 500, or 400 Torr.

In preferred embodiments, the vapor pressure of the primary modifier gasis at least 660 Torr at 37° C. and the vapor pressure of the gas osmoticagent is at least 100 Torr at 37° C. For in vivo imaging mean bubblediameters between 1 and 10 μm are preferred, with 3 to 5 μm mostpreferred. The invention may in one embodiment also be described as amixture of a first gas or gases and a second gas or gases withingenerally spherical membranes to form microbubbles, where the first gasand the second gas are respectively present in a molar ratio of about1:100, 1:75, 1:50, 1:30, 1:20, or 1:10 to about 1000:1, 500:1, 250:1,100:1, 75:1 or 50:1, and where the first gas has a vapor pressure of atleast about (760−x) mm Hg at 37° C., where x is the vapor pressure ofthe second gas at 37° C., and where the vapor pressure of each of thefirst and second gases is greater than about 75 or 100 mm Hg at 37° C.

Microbubbles prepared in accordance with one preferred embodiment of theinvention may also possess an additional advantageous property. In onesuch embodiment, mixtures of nonosmotic gases with osmotic stabilizinggases (or gas osmotic agents) are used to stabilize the resultant bubblesize distribution during and immediately after production. Upongeneration of the bubbles, the higher LaPlace pressure in smallerbubbles causes diffusion through the liquid phase to the lower LaPlacepressure larger bubbles. This causes the mean size distribution toincrease above the capillary dimension limit of 5 microns over time.This is called disproportionation. When a mixture of a nonosmotic gas(e.g., air) is used with an osmotic vapor (e.g., C₆ F₁₄) a slightreduction in volume of the smaller bubbles, due to air leaving thebubble, concentrates the osmotic gas and increases its osmotic pressurethus retarding further shrinkage while the larger bubbles increase involume slightly, diluting the osmotic gas and retarding further growth.

An additional advantage of using a mixture of an extremely blood solublegases (e.g., 87.5% by volume CO₂) and an osmotic gas mixture (e.g., 28%C₆ F₁₄ vapor+72% air) is that, when injected, these bubbles rapidlyshrink due to the loss of CO₂ to the blood. The bubbles, upon injection,will experience an 87.5% volume decrease due to loss of CO₂. This lossof CO₂ corresponds to a halving of the bubble diameter. Accordingly, onecan prepare larger diameter bubbles (e.g., 9 μm), using simplifiedmechanical means, that will shrink to below 5 microns upon injection. Ingeneral, such bubbles will initially be prepared where the first gas ispresent in a ratio of at least 1:1 with respect to the second gas,preferably at least 3:2, 2:1, 3:1, 4:1, 5:1, or 10:1. Where themicrobubble membrane is more permeable to the first gas than to thesecond gas (e.g., the membrane has respective permeabilities to thegases in a ratio of at least about 2:1, 3:1, 4:1, 5:1, or 10:1,preferably even higher, e.g., 20:1, 40:1, or 100:1), the bubblesadvantageously shrink from their original first diameter to an averagesecond diameter of 75% or less of their original diameter quite rapidly(e.g., within one, two, four, or five minutes). Then, when at least onerelatively membrane-permeable gas is present in the aqueous mediumsurrounding the microbubble, the bubble is preferably stabilized at orabout the second diameter for at least about 1 minute, preferably for 2,3, 4, or 5 minutes. In one preferred embodiment, the bubbles maintain asize between about 5 or 6 μm and 1 μm for at least 1, 2, 3, 4, or 5minutes, stabilized by a gas osmotic pressure differential. The gastension in the external liquid is preferably at least about 700 mm Hg.Moreover, a relatively membrane impermeable gas is also in themicrobubble to create such an osmotic pressure differential.

I. Microbubble Construction

A. The Membrane-Forming Liquid Phase

The external, continuous liquid phase in which the bubble residestypically includes a surfactant or foaming agent. Surfactants suitablefor use in the present invention include any compound or compositionthat aids in the formation and maintenance of the bubble membrane byforming a layer at the interface between the phases. The foaming agentor surfactant may comprise a single compound or any combination ofcompounds, such as in the case of co-surfactants.

Examples of suitable surfactants or foaming agents include: blockcopolymers of polyoxypropylene polyoxyethylene, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol,nonylphenoxy poly(ethyleneoxy)ethanol, derivatized starches, hydroxyethyl starch fatty acid esters, commercial food vegetable starches,dextrans, dextran fatty acid esters, sorbitol, sorbitol fatty acidesters, gelatin, serum albumins, and combinations thereof. Particularlypreferred sugar esters are those with a component having ahydrophilic-lipophilic balance (HLB) of less than 8 including mono-,di-, tri- or polyesterified sucrose, trehalose, dextrose and fructose toproduce stearate, behenate, palmitate, myristate, laurate and caproate(e.g. sucrose tristearate), as these sugar esters prolong microbubblestability. Other sugar esters contemplated for use in the presentinvention include those esterified with unsaturated fatty acids to formoleate, ricinoleate, linoleate, arachidate, palmitoleate andmyristoleate. The HLB is a number between 0 and 40 assigned toemulsifying agents and substances which are emulsified. The HLB isindicative of emulsification behavior and is related to the balancebetween the hydrophilic and lipophilic portions of the molecule (Rosen,M., (1989), Surfactants and Interfacial Phenomena, Second Edition, JohnWiley & Sons, New York, pp. 326-329).

In the present invention, preferred surfactants or foaming agents areselected from the group consisting of phospholipids, nonionicsurfactants, neutral or anionic surfactants, fluorinated surfactants,which can be neutral or anionic, and combinations of such emulsifying orfoaming agents.

The nonionic surfactants suitable for use in the present inventioninclude polyoxyethylene-polyoxypropylene copolymers. An example of suchclass of compounds is Pluronic, such as Pluronic F-68. Also contemplatedare polyoxyethylene fatty acids esters, such as polyoxyethylenestearates, polyoxyethylene fatty alcohol ethers, polyoxyethylatedsorbitan fatty acid esters, glycerol polyethylene glycol oxystearate,glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oils, and the hydrogenated derivatives thereof, andcholesterol. In addition, nonionic alkylglucosides such as Tweens®,Spans®, and Brijs® having a component with an HLB less than 8 are alsowithin the scope of the present invention. The Spans include sorbitantetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitantripalmitate, sorbitan trioleate, and sorbitan distearate. Tweensinclude polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitantripalmitate, polyoxyethylene sorbitan trioleate. The Brij family isanother useful category of materials, which includes polyoxyethylene 10stearyl ether. Anionic surfactants, particularly fatty acids (or theirsalts) having 12 to 24 carbon atoms, may also be used. One example of asuitable anionic surfactant is oleic acid, or its salt, sodium oleate.

It will be appreciated that a wide range of surfactants can be used.Indeed, virtually any surfactant or foaming agent (including those stillto be developed) capable of facilitating formation of the microbubblescan be used in the present invention. The optimum surfactant or foamingagent or combination thereof for a given application can be determinedthrough empirical studies that do not require undue experimentation.Consequently, one practicing the art of the present invention shouldchoose the surfactant or foaming agents or combination thereof basedupon such properties as biocompatibility or their non-Newtonianbehavior.

The blood persistence of a contrast agent is inversely proportional tothe LaPlace pressure which is proportional to the surface tension of thebubble. Reduced surface tension, therefore, increases blood persistence.Surfactants that form ordered structures (multilaminar sheets and rods)in solution and produce non-Newtonian viscoelastic surface tensions areespecially useful. Such surfactants include many of the sugar basedsurfactants and protein or glycoprotein surfactants (including bovine,human, or other lung surfactants). One preferred type of such surfactanthas a sugar or other carbohydrate head group, and a hydrocarbon orfluorocarbon tail group. A large number of sugars are known that canfunction as head groups, including glucose, sucrose, mannose, lactose,fructose, dextrose, aldose, and the like. The tail group preferably hasfrom about 2 or 4 to 20 or 24 carbon atoms, and may be, for example, afatty acid group (branched or unbranched, saturated or unsaturated)covalently bound to the sugar through an ester bond. The surface tensionof bubbles produced with these surfactants greatly decreases as thesurface is compressed by shrinkage of the bubble (e.g., when the bubbleshrinks), and it is increased as the surface area of the bubble isincreased (e.g., when the bubble grows). This effect retardsdisproportionation, which leads to narrower size distribution and longerpersisting bubbles in the vial and in vivo. A preferred surfactantmixture that has the properties associated with non-Newtonianviscoelasticity includes a nonionic surfactant or other foamingsurfactant in combination with one of the non-Newtonian viscoelasticsurfactant such as one of the sugar esters (e.g. 2% Pluronic F-68 plus1% sucrose stearate). Often the ratio of the nonionic surfactant to thenon-Newtonian surfactant is from about 5:1 to about 1:5, with thesurfactants together (whether non-Newtonian or more conventional)comprising 0.5 to 8%, more preferably about 1 to 5% (w/v) of themicrobubble-forming liquid mixture.

The lowering of surface tension in small bubbles, counter to typicalLaPlace pressure, allows the use of more efficient gas osmotic agentssuch as higher molecular weight perfluorocarbons as the gas osmoticagent. With conventional surfactants, the higher molecular weight PFCswill condense at the high bubble pressures. Without these efficientsurfactants higher boiling less membrane permeable PFCs, e.g. C₆ F₁₄,would be extremely difficult.

One may also incorporate other agents within the aqueous phase. Suchagents may advantageously include conventional viscosity modifiers,buffers such as phosphate buffers or other conventional biocompatiblebuffers or pH adjusting agents such as acids or bases, osmotic agents(to provide isotonicity, hyperosmolarity, or hyposmolarity). Preferredsolutions have a pH of about 7 and are isotonic. However, when CO₂ isused as a first gas in a bubble designed to shrink rapidly to a firstsize, a basic pH can facilitate rapid shrinkage by removing CO₂ as itleaves the bubble, preventing a buildup of dissolved CO₂ in the aqueousphase.

B. The Gas Phase

A major aspect of the present invention is in the selection of the gasphase. As was discussed above, the invention relies on the use ofcombinations of gases to harness or cause differentials in partialpressures and to generate gas osmotic pressures, which stabilize thebubbles. The primary modifier gas is preferably air or a gas present inair. Air and/or fractions thereof are also present in normal blood andserum. Where the microbubbles are to be used in an environment differentfrom blood, the primary modifier gas is preferably selected from gasesnormally present in the external medium. Another criteria is the easewith which the primary modifier gas is diffused into or out of thebubbles. Typically, air and/or fractions thereof are also readilypermeable through conventional flexible or rigid bubble surfaces. Thesecriteria, in combination, allow for the rapid diffusion of the primarymodifier gas into or out of the bubbles, as required.

Modifier gases not present in the external medium can also be used.However, in this case the bubble will initially grow or shrink(depending on the relative permeability and concentrations of theexternal gases to the modifier) as the external gases replace theoriginal modifier gas. If, during this process, the gas osmotic agenthas not condensed, the bubble will remain stable.

The gas osmotic agent is preferably a gas that is less permeable throughthe bubble's surface than the modifier. It is also preferable that thegas osmotic agent is less soluble in blood and serum. Therefore, it willnow be understood that the gas osmotic agent can be a gas at room orbody temperature or it can ordinarily be a liquid at body temperature,so long as it has a sufficient partial or vapor pressure at thetemperature of use to provide the desired osmotic effect.

Accordingly, fluorocarbons or other compounds that are not gases at roomor body temperature can be used, provided that they have sufficientvapor pressure, preferably at least about 50 or 100 Torr at bodytemperature, or more preferably at least about 150 or 200 Torr. Itshould be noted that where the gas osmotic agent is a mixture of gases,the relevant measure of vapor pressure is the vapor pressure of themixture, not necessarily the vapor pressure of the individual componentsof the mixed gas osmotic agent.

It is also important that where a perfluorocarbon is used as the osmoticagent within a bubble, the particular perfluorocarbon does not condenseat the partial pressure present in the bubble and at body temperature.Depending on the relative concentrations of the primary modifier gas andthe gas osmotic agent, the primary modifier gas may rapidly leave thebubble causing it to shrink and concentrate the secondary gas osmoticagent. Such shrinking may occur until the gas osmotic pressure equalsthe external pressure on the bubble (maximum absolute arterial pressure)plus the LaPlace pressure of the bubble minus the air tension, or airsaturation tension, of the blood (essentially one atmosphere). Thus thecondensing partial pressure of the resulting gas mixture at 37° C. mustbe above the equilibrium partial pressure, discussed above, of theosmotic agent.

Representative fluorocarbons meeting these criteria and in increasingability to stabilize microbubbles are as follows:CCl₂F₂<CF₄, CHClF₂<C₄F₁₀, N(C₂F₅)₃<C₅F₁₂<C⁻⁶F₁₄

Accordingly, it will be understood that PFC's with eight carbons atomsor fewer (37° C. vapor pressures greater than 80 mm Hg) are preferred.As will also be understood, however, it is possible to construct largermolecules with increased volatility through the addition of heteroatomsand the like. Therefore, the determination of the optimal secondary gasosmotic agent or gases agents is not size limited, but, rather, is basedupon its ability to retain its vapor phase at body temperature and whileproviding a gas osmotic pressure equal to at least the sum of thearterial and LaPlace pressures.

A listing of some compounds possessing suitable solubility and vaporpressure criteria is provided in Table I:

TABLE I perfluoro propanes, C₃ F₈ perfluoro butanes, C₄ F_(.10)perfluoro cyclo butanes, C₄ F_(.8) perfluoro pentanes, C₅ F₁₂ perfluorocyclo pentanes, C_(.5) F₁₀ perfluoro methylcyclobutanes, C₅ F₁₀perfluoro hexanes, C₆ F_(.4) perfluoro cyclohexanes, C₆ F₁₂ perfluoromethyl cyclopentanes, C₆ F₁₂ perfluoro dimethyl cyclobutanes, C₆F₁₂perfluoro heptanes, C₇ F_(.16) perfluoro cycloheptanes, C₇ F₁₄ perfluoromethyl cyclohexanes, C₇ F₁₄ perfluoro dimethyl cyclopentanes, C₇ F₁₄perfluoro trimethyl cyclobutanes, C₇ F₁₄ perfluoro triethylamines, N(C₂F₅)₃

It will be appreciated that one of ordinary skill in the art can readilydetermine other compounds that would perform suitably in the presentinvention that do not meet both the solubility and vapor pressurecriteria, described above. Rather, it will be understood that certaincompounds can be considered outside the preferred range in eithersolubility or vapor pressure, if such compounds compensate for theaberration in the other category and provide a superior insolubility orlow vapor pressure.

It should also be noted that for medical uses the gases, both themodifier gas and the gas osmotic agent, should be biocompatible or notbe physiologically deleterious. Ultimately, the microbubbles containingthe gas phase will decay and the gas phase will be released into theblood either as a dissolved gas or as submicron droplets of thecondensed liquid. It will be understood that gases will primarily beremoved from the body through lung respiration or through a combinationof respiration and other metabolic pathways in the reticuloendothelialsystem.

Appropriate gas combinations of the primary modifier and secondary gasescan be ascertained empirically without undue experimentation. Suchempirical determinations are described in the Examples.

When an efficient surfactant, e.g., bovine lung surfactant, is employedto produce a large diameter bubble with a low surface tension, theLaPlace pressure is very low. When perfluorooctylbromide (PFOB)saturated air is inside the bubble and the bubble is exposed to air or aliquid nearly saturated with air (e.g., equilibrated with air) the gasosmotic pressure is greater than the LaPlace pressure and therefore thebubble grows. With smaller diameter bubbles the LaPlace pressure ishigher and therefore the bubble shrinks and collapses. This shrinkage isat a reduced rate being driven by the difference between the LaPlacepressure and the gas osmotic pressure. When small diameter bubbles arecreated by sonicating gas or gas vapor mixtures in a low surface tensionsurfactant solution, e.g., 2% pluronic F-68 plus 1% sucrose stearate,the time the bubbles persist in vitro, as observed by microscope, and invivo as observed by Doppler ultrasound imaging of a rabbit's kidney postintravenous injection, correlated with the above gas osmotic pressurecomparison.

In the rabbit kidney Doppler experiment (Example III), contrastenhancement was observed for up to 10 minutes with perfluorohexane/airmixtures in the bubbles compared with the instantaneous disappearance ofcontrast with pure air microbubbles. Thus, these perfluorochemicals arecapable of exerting gas osmotic pressures that nearly counterbalance theLaPlace pressure and create functional ultrasound microbubble contrastagents.

A surprising discovery was that mixtures of PFCs, e.g., C₄ F₁₀ (as acombination modifier gas and a gas osmotic agent) saturated with C₆ F₁₄vapor (as the main gas osmotic agent), can stabilize the bubble forlonger times than either component alone. This is because C₄ F₁₀ is agas at body temperature (and, thus, can act as both a modifier gas and agas osmotic agent) has a somewhat reduced membrane permeability and itis only slightly soluble in C₆ F₁₄ at body temperature. In thissituation the gas osmotic pressures of both agents are added together,leading to increased bubble persistence over that of air/C₆ F₁₄ onlymixtures. It is possible that the condensing point of the longerpersisting higher molecular weight C₆ F₁₄ component is increased,allowing a larger maximum gas osmotic pressure to be exerted. Othermixtures of PFCs will perform similarly. Preferred mixtures of PFCs willhave ratios of 1:10 to 10:1, and include such mixtures asperfluorobutane/perfluorohexane and perfluorobutane/perfluoropentane.These preferred fluorochemicals can be branched or straight chain.

As was discussed above, we have also discovered that mixtures ofnonosmotic gases in combination with the gas osmotic agent act tostabilize the size distribution of the bubbles before and afterinjection. Upon generation of the bubbles, the higher LaPlace pressuresin smaller bubbles causes diffusion through the liquid phase to thelower LaPlace pressure larger bubbles. This causes the mean sizedistribution to increase above the capillary dimension limit of 5microns with time. This is called disproportionation.

However, when a mixture of a modifier gases (e.g., air or carbondioxide) are used with a gas osmotic agent (e.g., C₆ F₁₄) a slightreduction in volume of the smaller bubbles, due to one of the modifiergases leaving the bubble, will concentrate the osmotic gas and increasesits osmotic pressure, thus, retarding further shrinkage. On the otherhand, the larger bubbles will increase in volume slightly, diluting theosmotic gas and also retarding further growth.

An additional advantage of using a mixture of an extremely blood solublegas (e.g., 75% through 87.5% by volume CO₂) and an osmotic gas mixture(e.g. 28% C₆ F₁₄ vapor and 72% air) is that when injected, these bubblesrapidly shrink due to the loss of CO₂ to the blood. Carbon dioxideleaves particularly fast due to a specific plasma enzyme that catalyzesits dissolution. An 87.5% volume decrease due to loss of CO₂ correspondswith a halving of the bubble diameter. Accordingly, larger diameterbubbles can be produced which will shrink to an appropriate size (i.e.,5 microns) upon injection or exposure to a solution with a basic oralkaline pH.

Accordingly, we have discovered that through use of a gas that isrelatively hydrophobic and that has a relatively low membranepermeability, the rate of contrast particle decay can be reduced. Thus,through reducing the particle decay rate, the microbubbles' half livesare increased and contrast enhancement potential is extended.

II. Other Components.

It will be understood that other components can be included in themicrobubble formulations of the present invention. For example, osmoticagents, stabilizers, chelators, buffers, viscosity modulators, airsolubility modifiers, salts, and sugars can be added to fine tune themicrobubble suspensions for maximum life and contrast enhancementeffectiveness. Such considerations as sterility, isotonicity, andbiocompatibility may govern the use of such conventional additives toinjectable compositions. The use of such agents will be understood tothose of ordinary skill in the art and the specific quantities, ratios,and types of agents can be determined empirically without undueexperimentation.

III. Formation of the Microbubbles of the Present Invention.

There are a variety of methods to prepare microbubbles in accordancewith the present invention. Sonication is preferred for the formation ofmicrobubbles, i.e., through an ultrasound transmitting septum or bypenetrating a septum with an ultrasound probe including anultrasonically vibrating hypodermic needle. However, it will beappreciated that a variety of other techniques exist for bubbleformation. For example, gas injection techniques can be used, such asventuri gas injection.

Other methods for forming microbubbles include formation of particulatemicrospheres through the ultrasonication of albumin or other protein asdescribed in European Patent Application 0,359,246 by MolecularBiosystems, Inc.; the use of tensides and viscosity increasing agents asdescribed in U.S. Pat. No. 4,446,442; lipid coated, non-liposomal,microbubbles as is described in U.S. Pat. No. 4,684,479; liposomeshaving entrapped gases as is described in U.S. Pat. Nos. 5,088,499 and5,123,414; and the use of denatured albumin particulate microspheres asis described in U.S. Pat. No. 4,718,433. The disclosure of each of theforegoing patents and applications is hereby incorporated by reference.

Any of the above methods can be employed with similar success to entrainthe modifier gases and gas osmotic agents of the present invention.Moreover, it is expected that similar enhancement in longevity of thebubbles created will be observed through use of the invention.

Sonication can be accomplished in a number of ways. For example, a vialcontaining a surfactant solution and gas in the headspace of the vialcan be sonicated through a thin membrane. Preferably, the membrane isless than about 0.5 or 0.4 mm thick, and more preferably less than about0.3 or even 0.2 mm thick, i.e., thinner than the wavelength ofultrasound in the material, in order to provide acceptable transmissionand minimize membrane heating. The membrane can be made of materialssuch as rubber, Teflon, mylar, urethane, aluminized film, or any othersonically transparent synthetic or natural polymer film or film formingmaterial. The sonication can be done by contacting or even depressingthe membrane with an ultrasonic probe or with a focused ultrasound“beam.” The ultrasonic probe can be disposable. In either event, theprobe can be placed against or inserted through the membrane and intothe liquid. Once the sonication is accomplished, the microbubblesolution can be withdrawn from a vial and delivered to the patient.

Sonication can also be done within a syringe with a low powerultrasonically vibrated aspirating assembly on the syringe, similar toan inkjet printer. Also, a syringe or vial may be placed in andsonicated within a low power ultrasonic bath that focuses its energy ata point within the container.

Other types of mechanical formation of microbubbles are alsocontemplated. For example, bubbles can be formed with a mechanical highshear valve (or double syringe needle) and two syringes, or an aspiratorassembly on a syringe. Even simple shaking may be used. The shrinkingbubble techniques described herein are particularly suitable formechanically formed bubbles, having lower energy input than sonicatedbubbles. Such bubbles will typically have a diameter much larger thanthe ultimately desired biocompatible imaging agent, but can be made toshrink to an appropriate size in accordance with the present invention.

In another method, microbubbles can be formed through the use of aliquid osmotic agent emulsion supersaturated with a modifier gas atelevated pressure introduced into in a surfactant solution. Thisproduction method works similarly to the opening of soda pop, where thegas foams upon release of pressure forming the bubbles.

In another method, bubbles can be formed similar to the foaming ofshaving cream, where perfluorobutane, freon, or another like materialthat boils when pressure is released. However, in this method it isimperative that the emulsified liquid boils at sufficiently lowtemperatures or that it contain numerous bubble nucleation sites so asto prevent superheating and supersaturation of the aqueous phase. Thissupersaturation will lead to the generation of a small number of largebubbles on a limited number of nucleation sites rather than the desiredlarge number of small bubbles (one for each droplet).

In still another method, dry void-containing particles or otherstructures (such as hollow spheres or honeycombs) that rapidly dissolveor hydrate, preferably in an aqueous solution, e.g., albumin, microfinesugar crystals, hollow spray dried sugar, salts, hollow surfactantspheres, dried porous polymer spheres, dried porous hyaluronic acid, orsubstituted hyaluronic acid spheres, or even commercially availabledried lactose microspheres can be stabilized with a gas osmotic agent.

For example, a spray dried surfactant solution can be formulated byatomizing a surfactant solution into a heated gas such as air, carbondioxide, nitrogen, or the like to obtain dried 1-10 micron or largerhollow or porous spheres, which are packaged in a vial filled with anosmotic gas or a desired gas mixture as decribed herein. The gas willdiffuse into the voids of the spheres. Diffusion can be aided bypressure or vacuum cycling. When reconstituted with a sterile solutionthe spheres will rapidly dissolve and leave osmotic gas stabilizedbubbles in the vial. In addition, the inclusion of starch or dextrins, asugar polyester and/or an inflating agent such as methylene chloride,1,1,2-trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, N.J.)or perfluorohexane, will result in microbubbles with an increased invivo half-life. Particularly preferred starches for use in formation ofmicrobubbles include those with a molecular weight of greater than about500,000 daltons or a dextrose equivalency (DE) value of less than about12. The DE value is a quantitative measurement of the degree of starchpolymer hydrolysis. It is a measure of reducing power compared to adextrose standard of 100. The higher the DE value, the greater theextent of starch hydrolysis. Such preferred starches include food gradevegetable starches of the type commercially available in the foodindustry, including those sold under the trademark N-LOK and CAPSULE byNational Starch and Chemical Co., (Bridgewater, N.J.); derivatizedstarches such as hydroxethyl starch (available under the trademarksHETASTARCH and HESPAN from du Pont Pharmaceuticals)(M-Hydroxyethylstarch, Ajinimoto, Tokyo, Japan). (Note that short chainstarches spray dry well and can be used to produce microbubbles, but arenot preferred because those with a molecular weight less than about500,000 do not stabilize the microbubbles. However, they can be used inthe present invention in applications in which additional stabilizationis not required.) In the alternative, a lyophilized cake of surfactantand bulking reagents produced with a fine pore structure can be placedin a vial with a sterile solution and a head spaced with an osmotic gasmixture. The solution can be frozen rapidly to produce a fine icecrystal structure and, therefore, upon lyophilization produces the finepores (voids where the ice crystals were removed.)

Alternatively, any dissolvable or soluble void-forming structures may beused. In this embodiment, where the void-forming material is not madefrom or does not contain surfactant, both surfactant and liquid aresupplied into the container with the structures and the desired gas orgases. Upon reconstitution these voids trap the osmotic gas and, withthe dissolution of the solid cake, form microbubbles with the gas orgases in them.

It will be appreciated that kits can be prepared for use in making themicrobubble preparations of the present invention. These kits caninclude a container enclosing the gas or gases described above forforming the microbubbles, the liquid, and the surfactant. The containercan contain all of the sterile dry components, and the gas, in onechamber, with the sterile aqueous liquid in a second chamber of the samecontainer. Suitable two-chamber vial containers are available, forexample, under the trademarks WHEATON RS177FLW or S-1702FL from WheatonGlass Co., (Millville, N.J.). Such a container is illustrated in FIGS.1-4. Referring to FIGS. 1 and 2, the illustrated Wheaton container 5 hasan upper chamber 20 which can contain an aqueous solution 25, and alower chamber 30 which can contain the dried ingredients 35 and adesired gas. A stopper 10 is provided separating the top chamber fromthe environment, and a seal 15 separates the upper chamber 20 from thelower chamber 30 containing spray dried (powdered) hollow microsphere 35and the gas osmotic agent. Depressing the stopper 10 pressurizes therelatively incompressible liquid, which pushes the seal 15 downward intothe lower chamber 30. This releases aqueous solution 25 into lowerchamber 30, resulting in the dissolution of powder 35 to form stabilizedmicrobubbles 45 containing the entrapped gas osmotic agent. Excess gasosmotic agent 40 is released from the lower chamber 30 into the upperchamber 20. This arrangement is convenient for the user and has theadded unexpected advantage of sealing the small quantity ofwater-impermeable gas osmotic agent in the lower chamber by covering theinterchamber seal with a thick (0.5 to 1.25 inch) layer of aqueoussolution and the advantage that the aqueous solution can be introducedinto the lower chamber without raising the pressure in the powderchamber by more than about 10%. Thus, there is no need for a pressurevent. (In contrast, conventional reconstitution of a solute in a singlechamber vial with a needle and syringe without a vent can result in theproduction of considerable intrachamber pressure which could collapsethe microbubbles.) The formation of microbubbles by this method isdescribed in Example XIII.

Alternatively, an inverted two-chamber vial may be used for microbubblepreparation. Referring to FIGS. 3 and 4, the same vial is used asdescribed hereinabove, except that the stopper 50 is elongated such thatit dislodges inner seal 15 when depressed. In this microbubblepreparation method, the spray dried hollow microspheres 35 and the gasosmotic agent 40 are contained in the upper chamber 20. The aqueoussolution 25 and gas osmotic agent 40 are contained within lower chamber30. When stopper 50 is depressed, it dislodges seal 15 allowing thespray dried hollow microspheres to mix with the aqueous solution 25 inthe presence of gas osmotic agent 40. Onc advantage associated with thismethod of microbubble formation is that the aqueous phase can beinstilled first and sterilized via autoclaving or other means, followedby instillation of the spray dried microspheres. This will preventpotential microbial growth in the aqueous phase prior to sterilization.

Although one particular dual chamber container has been illustrated,other suitable devices are known and are commercially available. Forexample, a two compartment glass syringe such as the B-D HYPAKLiquid/Dry 5+5 ml Dual Chamber prefilled syringe system (BectonDickinson, Franklin Lakes, N.J.; described in U.S. Pat. No. 4,613,326)can advantageously be used to reconstitute the spray dried powder. Theadvantages of this system include:

-   -   1. Convenience of use;    -   2. The aqueous-insoluble gas osmotic agent is sealed in by a        chamber of aqueous solutin on one side and an extremely small        area of elastomer sealing the needle on the other side; and    -   3. A filtration needle such as Monoject #305 (Sherwood Medical,        St. Louis, Mo.) can be fitted onto the syringe at the time of        manufacture to ensure that no undissolved solids are injected.

The use of the two chamber syringe to form microbubbles is described inExample XIV.

It can be appreciated by one of ordinary skill in the art that othertwo-chamber reconstitution systems capable of combining the spray driedpowder with the aqueous solution in a sterile manner are also within thescope of the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble osmotic gas and the environment, to increase shelf lifeof the product.

Alternatively, the container can contain the void forming material andthe gas or gases, and the surfactant and liquid can be added to form themicrobubbles. In one embodiment, the surfactant can be present with theother materials in the container, so that only the liquid needs to beadded in order to form the microbubbles. Where a material necessary forforming the microbubbles is not already present in the container, it canbe packaged with the other components of the kit, preferably in a formor container adapted to facilitate ready combination with the othercomponents of the kit.

The container used in the kit may be of the type described elsewhereherein. In one embodiment, the container is a conventional septum-sealedvial. In another, it has a means for directing or permitting applicationof sufficient bubble forming energy into the contents of the container.This means can comprise, for example, the thin web or sheet describedpreviously.

Various embodiments of the present invention provide surprisingadvantages. The spray dried starch formulations give prolonged in-vialstability of the microbubbles, particularly when the molecular weight ofthe starch is over about 500,000. Use of fatty acid esters of sugarscontaining a component with an HLB of less than 8 provides greatlyincreased in vivo and in-vial stability. Fatty acid esters of sugarssuch as sucrose monosterate, as well as block copolymers such PluronicF-68 (with an HLB over 12) allow the powder to form bubbles at theinstant they are rehydrated. Spray dried formulations with a structuralagent such as a starch, starch derivative, or dextrin provide asignificantly lower total dose of surfactant than comparable sonicatedformulations. The use of two-chamber vials with water providing anadditional seal for the fluorocarbon gas provide increased shelf life,and greater use convenience. Spray dried formulations with a structuralagent (such as a starch or dextrin) and a sugar polyester providemicrobubbles with greatly increased in vivo half lives.

The inclusion of an inflating agent in the solution to be spray-driedresults in a greater ultrasound signal per gram of spray-dried powder byforming a greater number of hollow microspheres. The inflating agentnucleates steam bubble formulation within the atomized droplets of thesolution entering the spray dryer as these droplets mix with the hot airstream within the dryer. Suitable inflating agents are those thatsupersaturate the solution within the atomized droplets with gas orvapor, at the elevated temperature of the drying droplets (approximately100° C.). Suitable agents include:

-   -   1. Dissolved low-boiling (below 100° C.) solvents with limited        miscibility with aqueous solutions, such as methylene chloride,        acetone and carbon disulfide used to saturate the solution at        room temperature.    -   2. A gas, e.g. CO₂ or N₂, used to saturate the solution at room        temperature and elevated pressure (e.g. 3 bar). The droplets are        then supersaturated with the gas at 1 atmosphere and 100° C.    -   3. Emulsions of immiscible low-boiling (below 100° C.) liquids        such as Freon 113, perfluoropentane, perfluorohexane,        perfluorobutane, pentane, butane, FC-11, FC-11B1, FC-11B2,        FC-12B2, FC-21, FC-21B1, FC-21B2, FC-31B1, FC-113A, FC-122,        FC-123, FC-132, FC-133, FC-141, FC-141B, FC-142, FC-151, FC-152,        FC-1112, FC-1121 and FC-1131.

The inflating agent is substantially evaporated during the spray dryingprocess and thus is not present in the final spray-dried powder in morethan trace quantities.

The inclusion of the surfactants and wetting agents into the shell ofthe microsphere allows the use of a lower surfactant concentration. Asthe microsphere shell is dissolving, it temporarily surrounds themicrobubble formed in its interior with a layer of aqueous phase that issaturated with the surfactants, enhancing their deposition on themicrobubble's surface. Thus, spray-dried surfactant containingmicrospheres require only locally high concentrations of surfactant, andobviate the need for a high surfactant concentration in the entireaqueous phase.

Any of the microbubble preparations of the present invention may beadministered to a vertebrate, such as a bird or a mammal, as a contrastagent for ultrasonically imaging portions of the vertebrate. Preferably,the vertebrate is a human, and the portion that is imaged is thevasculature of the vertebrate. In this embodiment, a small quantity ofmicrobubbles (e.g., 0.1 ml/Kg [2 mg/Kg spray-dried powder] based on thebody weight of the vertebrate) is introduced intravascularly into theanimal. Other quantities of microbubbles, such as from about 0.005 ml/Kgto about 1.0 ml/Kg, can also be used. Imaging of the heart, arteries,veins, and organs rich in blood, such as liver, lungs, and kidneys canbe ultrasonically imaged with this technique.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, exemplary ofpreferred methods of practicing the present invention and are notlimiting of the scope of the invention or the claims appended hereto.

EXAMPLE I Preparation of Microbubbles Through Sonication

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants, air as a modifiergas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. Air saturated with perfluorohexane vapor (220 torr ofperfluorohexane with 540 torr of air) at 25° C. was used to flush theheadspace of the vial. The vial was sealed with a thin 0.22 mmpolytetrafluoroethylene (PTFE) septum. The vial was turned horizontally,and a ⅛″ (3 mm) sonication probe attached to a 50 watt sonicator modelVC50, available from Sonics & Materials was pressed gently against theseptum. In this position, the septum separates the probe from thesolution. Power was then applied to the probe and the solution wassonicated for 15 seconds, forming a white solution of finely dividedmicrobubbles, having an average number weighted size of 5 microns asmeasured by Horiba LA-700 laser light scattering particle analyzer.

EXAMPLE II Measurement of In-Vitro Size of Microbubbles

The in-vitro size of the microbubbles prepared in Example I was measuredby laser light scattering. Studies of bubbles were conducted where themicrobubbles were diluted into a 4% dextrose water solution (1:50)circulating through a Horiba LA-700 laser light scattering analyzer. Theaverage microbubbles size was 5 microns and doubled in size in 25minutes.

Interestingly, microbubbles prepared through the same method in ExampleI without the use of a gas osmotic agent (substituting air for theperfluorohexane/air mixture) had an average size of 11 microns and gaveonly background readings on the particle analyzer at 10 seconds.

EXAMPLE III Measurement of In-Vivo Lifetime of Microbubbles

The lifetimes of microbubbles prepared in accordance with Example I weremeasured in rabbits through injecting 0.2 ml of freshly formedmicrobubbles into the marginal ear vein of a rabbit that was underobservation with a Accuson 128XP/5 ultrasound imaging instrument with a5 megahertz transducer. Several tests were conducted, during whichimages of the heart, inferior vena cava/aorta, and kidney were obtainedwhile measuring the time and extent of the observable contrastenhancement. The results are presented in the following Table II:

TABLE II TIME TO TIME MINIMUM TO NO TIME MAX. USABLE ENHANCE- ORGAN DOSEINTENSITY INTENSITY MENT Heart 0.1 ml/Kg 7-10 sec. 8-10 min. 25 minIVC/Aorta 0.1 ml/Kg 7-10 sec. 8-10 min. 25 min Kidney 0.1 ml/Kg 7-10sec. 8-10 min. 25 min

In Table III, a comparison of microbubbles prepared in an identicalfashion without the use of an osmotic gas is presented (only air wasused). Note that sporadic reflections were observed only in the rightheart ventricle during the injection but disappeared immediately postdosing.

TABLE III TIME TO TIME TIME TO MINIMUM TO NO MAXIMUM USABLE ENHANCE-ORGAN DOSE INTENSITY INTENSITY MENT Heart 0.1 ml/Kg 0 0 0 IVC/Aorta 0.1ml/Kg 0 0 0 Kidney 0.1 ml/Kg 0 0 0

The use of an osmotic or gas osmotic agent dramatically increased thelength of time for which microbubbles are visible.

EXAMPLE IV Preparation of Mixed Osmotically Stabilized Microbubbles

Microbubbles with an average number weighted size of 5 microns wereprepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants and mixtures ofperfluorohexane and perfluorobutane as the gas osmotic agents.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl and 2% Pluronic F-68 was added to a 2.0 ml vial. The vial had aremaining head space of 0.7 ml, initially containing air. An osmotic gasmixture of perfluorohexane, 540 Torr and perfluorobutane at 220 Torr wasused to flush the headspace before sealing with a thin 0.22 mm PTFEseptum. The vial was sonicated as in Example I, forming a white solutionof finely divided microbubbles, having an average particle size of 5microns as measured by a Horiba LA-700 laser light scattering particleanalyzer. This procedure was repeated twice more, once with pureperfluorobutane and then with a 540 Torr air+220 Torr perfluorohexanemixture. Vascular persistence of all three preparations was determinedby ultrasound imaging of a rabbit post I.V. injection and are listedbelow:

1.5 minutes perfluorobutane 2 minutes perfluorohexane + air 3 minutesperfluorbutane + perfluorohexane

The mixture of perfluorocarbons persisted longer than either agentalone.

EXAMPLE V Preparation of Gas Osmotically Stabilized Microbubbles fromSoluble Spray Dried Spheres

Gas osmotically stabilized microbubbles were prepared by dissolvinghollow spray dried lactose spheres, filled with an air perfluorohexanevapor mixture, in a surfactant solution.

Spray dried spheres of lactose with a mean diameter of approximately 100micron and containing numerous 10 to 50 micron cavities, was obtainedfrom DMV International under the trade name of Pharmatose DCL-11. Ninetymilligrams of the lactose spheres was placed in a 2.0 ml vial. Theporous spheres were filled with a mixture of 220 Torr perfluorohexaneand 540 Torr air by cycling the gas pressure in the vial between oneatmosphere and ½ atmosphere a total of 12 times over 5 minutes. Asurfactant solution containing 0.9% sodium chloride, 2% Pluronic-F₆₈ and1% sucrose stearate was warmed to approximately 45° C., to speed thedissolution of the lactose, before injecting 1.5 ml of the warmedsolution into the vial. The vial was then gently agitated by inversionfor approximately 30 seconds to dissolve the lactose before injectingthe microbubbles thus prepared into the Horiba LA-700 particle analyzer.A 7.7 micron volume weighted median diameter was measured approximatelyone minute after dissolution. The diameter of these microbubblesremained nearly constant, changing to a median diameter of 7.1 micronsin 10 minutes. When the experiment was repeated with air filled lactose,the particle analyzer gave only background readings one minute afterdissolution, thus demonstrating that gas osmotically stabilizedmicrobubbles can be produced by the dissolution of gas-filledcavity-containing structures.

EXAMPLE VI Preparation of Larger Bubbles that Shrink to a Desired Size

Microbubbles with an average volume weighted size of 20 micronsshrinking to 2 microns were prepared by sonication of an isotonicaqueous phase containing 2% Pluronic F-68 as the surfactant, CO₂ as adiluent gas and perfluorohexane as the gas osmotic agent.

In this experiment, 1.3 ml of a sterile water solution containing 0.9%NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 mlvial. The vial had a remaining head space of 0.7 ml initially containingair. A mixture of air saturated with perfluorohexane at 25° C. dilutedby a factor of 10 with CO₂ (684 Torr CO₂+54 Torr air+22 Torrperfluorohexane) was used to flush the head space. The vial was sealedwith a thin 0.22 mm PTFE septum. The vial was sonicated as in Example I,forming a white solution of finely divided microbubbles, having anaverage particle size of 28 microns as measured by Horiba LA-700 laserlight scattering analyzer. In the 4% dextrose+0.25 mM NaOH solution ofthe Horiba, the average bubble diameter rapidly shrank in 2 to 4 minutesfrom 28 microns to 5 to 7 microns, and then remained relativelyconstant, reaching 2.6 micron after 27 minutes. This is because the CO₂leaves the microbubbles by dissolving into the water phase.

EXAMPLE VII Perfluoroheptane Stabilized Microbubble In Vitro Experiment

Microbubbles were prepared as in Example I above employingperfluoroheptane saturated air (75 torr plus 685 torr air) and weremeasured as in Example II above. The average number weighted diameter ofthese microbubbles was 7.6 micron, one minute after circulation, and 2.2microns after 8 minutes of circulation. This persistence, compared tothe near immediate disappearance of microbubbles containing only air,demonstrates the gas osmotic stabilization of perfluoroheptane.

EXAMPLE VIII Perfluorotripropyl Amine Stabilized Microbubble In vivoExperiment

Microbubbles were prepared as in Example I above, employingperfluorotripropyl amine saturated air and were assessed as in ExampleIII above. The usable vascular persistence of these microbubbles wasfound to be 2.5 minutes, thus demonstrating the gas osmoticstabilization of perfluorotripropyl amine.

EXAMPLE IX Effect of a Non Newtonian Viscoelastic Surfactant-SucroseStearate

Microbubbles were prepared as in Example I above employing 0.9% NaCl, 2%Pluronic F-68 and 2% sucrose stearate as the surfactant and withperfluoropropane saturated air and perfluorohexane saturated air in theheadspace. These two preparations were repeated with the same surfactantsolution minus sucrose stearate. All four microbubble preparations wereassessed as in Example III above. The usable vascular persistence ofthese microbubbles are listed below:

-   -   2% Pluronic F-68+2% sucrose stearate persistence        -   2 minutes perfluoropropane        -   4 minutes perfluorohexane    -   2% Pluronic F-68 only persistence        -   2 minutes perfluoropropane        -   1 minute perfluorohexane

As seen above, the reduced surface tension made possible by thenon-Newtonian viscoelastic properties of sucrose stearate prevented theless volatile perfluorohexane from condensing, allowing perfluorohexanemicrobubbles of longer persistence to be produced.

EXAMPLE X Spray Drying of Starch-containing Emulsion

One liter of each of the following solutions was prepared with water forinjection: Solution A containing 4.0% w/v N-Lok vegetable starch(National Starch and Chemical Co., Bridgewater, N.J.) and 1.9% w/vsodium chloride (Mallinckrodt, St. Louis, Mo.) and Solution B containing2.0% Superonic F-68 (Serva, Heidelberg, Germany) and 2.0% w/v RyotoSucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, Japan).Solution B was added to a high shear mixer and cooled in an ice bath. Acoarse suspension of 40 ml 1,1,2-trichlorotrifluoroethane (Freon 113; EMScience, Gibbstown, N.J.) was made in the 1 liter of solution B. Thissuspension was emulsified using a Microfluidizer (MicrofluidicsCorporation, Newton, Mass.; model M-110F) at 10,000 psi, 5° C. for 5passes. The resulting emulsion was added to solution A to produce thefollowing formula for spray drying:

-   -   2.0% w/v N-Lok    -   1.0% w/v Superonic F-68    -   1.0% w/v Sucrose Stearate S-1670    -   0.95% w/v sodium chloride    -   2.0% v/v 1,1,2-trichlorotrifluoroethane

This emulsion was then spray dried in a Niro Atomizer Portable SprayDryer equipped with a two fluid atomizer (Niro Atomizer, Copenhagen,Denmark) employing the following settings:

-   -   hot air flow rate=39.5 CFM    -   inlet air temp.=235° C.    -   outlet air temp.=10° C.    -   atomizer air flow=110 liters/hr    -   emulsion feed rate=liter/hr

The dry, hollow spherical product had a diameter between about 1 μm andabout 15 μm and was collected at the cyclone separator as is standardfor this dryer. Aliquots of powder (250 mg) were weighed into 10 mltubing vials, sparged with perfluorohexane-saturated nitrogen at 13° C.and sealed. The nitrogen was saturated with perfluorohexane by passingit through three perfluorohexane filled gas washing bottles immersed ina 13° C. water bath.

The vials were reconstituted with 5 ml water for injection afterinserting an 18-gauge needle as a vent to relieve pressure as the waterwas injected. One ml of the resulting microbubble suspension wasinjected intravenously into an approximately 3 kg rabbit instrumented tomonitor the Doppler ultrasound signal of its carotid artery. A 10 MHzflow cuff (Triton Technology Inc., San Diego, Calif.; model ES-10-20)connected to a System 6 Doppler flow module (Triton Technology Inc.) fedthe RF doppler signal to a LeCroy 9410 oscilloscope (LeCroy, ChestnutRidge, N.Y.). The root mean square (RMS) voltage of the signal computedby the oscilloscope was transferred to a computer and the resultantcurve fitted to obtain peak echogenic signal intensity and half-life ofthe microbubbles in blood. Signals before contrast were less than 0.1volts RMS. A peak signal of 1.6 volts RMS was observed with a half lifeof 52.4 seconds, thus demonstrating the ability of spray dried powdersto produce intravascular echogenic microbubbles that have a prolongedhalf-life in vivo.

EXAMPLE XI Production of Microbubbles Containing Hydroxyethyl Starch

An emulsion for spray drying was prepared as in Example X to give afinal composition of:

-   -   2.0% w/v m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan)    -   2.0% w/v sodium chloride (Mallinckrodt)    -   1.74% sodium phosphate, dibasic (Mallinckrodt)    -   0.26% w/v sodium phosphate, monobasic (Mallinckrodt)    -   1.7% w/v Superonic F-68 (Serva)    -   0.2% w/v Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food        Corp.)    -   0.1% w/v Ryoto Sucrose Stearate S-570 (Mitsubishi-Kasei Food        Corp.)    -   4.0% w/v 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science,        Gibbstown, N.J.)

This emulsion was spray dried as in Example X using the followingparameters:

-   -   hot air flow rate=39.5 CFM    -   inlet air temperature=220° C.    -   outlet air temperature=105° C.    -   atomizer air flow=110 liter/hr    -   emulsion feed rate=1 liter/hr

Sample vials were prepared with 400 mg spray dried powder and sparged asin Example X. After reconstitution with 5 ml water for injection andintravenous administration of 1.0 ml to a rabbit as in Example X, a peaksignal of 2.4 volts RMS was observed with a half life of 70.9 seconds.This demonstrates the ability of higher molecular weight and derivatizedinjectable grade starches and sugar polyesters to produce higher in vivosignals which persist longer as compared to standard vegetable starch(Example X). This also demonstrates the lower surfactant concentrationrequired by an optimal spray dried formula and the use of a spray dryinginflating agent such as Freon 113.

EXAMPLE XII Stability of Starch-Containing Microbubbles

The spray dried powder described in Example X was reconstituted asdescribed. For comparison, the sonicated microbubbles from Example Iwere prepared and both were examined by light microscopy within 2minutes of preparation and again at 10 minutes after preparation. Thesonicated product was found to have a wider initial bubble diameterdistribution (between about 1 and about 30 μm) than the reconstitutedspray dried powder (between about 3 and about 15 μm). After 10 minutes,the vials were agitated, resampled and again observed by lightmicroscopy. The reconstituted spray dried product was essentiallyunchanged, while the sonicated microbubbles had grown bydisproportionation until nearly all of the observable microbubbles weremore than 10 μm in diameter. This experiment demonstrates the extendedin-vial stability of microbubbles produced by spray drying and theability of starches to increase in vitro stability.

EXAMPLE XIII Microbubble Formation Using Two Chamber Vial

800 mg spray dried powder from Example XII were weighed into the lowerchamber of a 20 ml Wheaton RS-177FLW two chamber vial (FIG. 1). The vialwas flushed with perfluorohexane-saturated nitrogen at 13° C. beforeinserting the interchamber seal. The upper chamber was filled with 10 mlsterile water for injection. The upper chamber stopper was inserted soas to eliminate all air bubbles in the upper chamber. Upon depression ofthe upper stopper, the interchamber seal was forced into the lowerchamber, allowing the water to flow into the lower chamber andreconstitute the powder (FIG. 2). Numerous stable microbubbles wereformed as demonstrated by light microscopy. This procedure demonstratesthe convenience of this form of packaging and the elimination of theneed to provide a vent to eliminate pressure buildup when the aqueousphase is added to the powder.

EXAMPLE XIV Microbubble Formation Using Two Chamber Syringe

One hundred mg of the spray dried powder from Example XI was weighedinto a 5 ml+5 ml HYPAK Liquid/Dry dual chamber syringe (BectonDickinson, Franklin Lakes, N.J.) and shaken into the powder (needle end)chamber. The interchamber seal was then positioned just above the bypasschannel. A 5 μm filter-containing needle was then fitted on the syringe.The powder-containing chamber was then filled with the gas osmotic agentby placing the assembly in a vacuum chamber, evacuating and refillingthe chamber with the gas osmotic agent, perfluorohexane-saturatednitrogen at 13° C. The filter needle allows the evacuation and refillingof the atmosphere in the powder-containing chamber. A sealing needlecover was then placed on the needle. The liquid chamber was then filledwith 4 ml water for injection and the plunger was seated using atemporary vent (wire inserted between the glass syringe barrel and theplunger so as to eliminate all air bubbles.)

To reconstitute, the needle sealing cover was removed to eliminatepressure buildup in the powder chamber. The plunger was then depressed,forcing the interchamber seal to the bypass position which allowed thewater to flow around the interchamber seal into the powder-containingchamber. The plunger motion was stopped when all the water was in thepowder chamber. The syringe was agitated to dissolve the powder. Excessgas and any large bubbles were expelled by holding the syringe, needleend up, and further depressing the plunger. The solution containingnumerous stabilized microbubbles (as observed by light microscopy) wasthen expelled from the syringe by depressing the plunger to its limit.

The foregoing description details certain preferred embodiments of thepresent invention and describes the best mode contemplated. It will beappreciated, however, that no matter how detailed the foregoing appearsin text, the invention can be practiced in many ways and the inventionshould be construed in accordance with the appended claims and anyequivalents thereof.

1. A method of forming stabilized microbubbles for in vivo use,comprising the steps of: providing a first gas, a second gas comprisinga perfluorocarbon, a membrane forming material, and a liquid, whereinsaid first gas and said second gas are present in a molar ratio of about1:100 to about 1,000:1, and wherein said second gas is a gas at37.degree. C. and 760 mm Hg with the proviso that said first gas andsaid second gas are not water vapor; and surrounding said first gas andsaid second gas with said membrane forming material to form stabilizedmicrobubbles in said liquid.
 2. The method of claim 1, wherein themembrane forming material comprises at least one surfactant.
 3. Themethod of claim 1, wherein the membrane forming material comprises aprotein.
 4. The method of claim 1 wherein the second gas is a gasosmotic agent.
 5. The method of claim 1 wherein the first gas is amodifier gas.
 6. The method of claim 4 wherein the second gas isselected from the group consisting of perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoromethylcyclobutane, perfluoropentane,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclobutanes, perfluorohexane, perfluorocyclohexane,perfluoroheptane, perfluorocycloheptane, perfluoromethylcyclohexane,perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane, andperfluorotriethylaxnine.
 7. The method of claim 1 wherein the first gasis selected from the group consisting of nitrogen, oxygen, carbondioxide, air and mixtures thereof.
 8. The method of claim 1 wherein theliquid is water.
 9. The method of claim 1 wherein the microbubbles areosmotically stabilized after being injected in vivo.
 10. The method ofclaim 3 wherein the protein is albumin.
 11. The method of claim 1wherein the second gas is perfluorohexane and the modifier first gas isnitrogen.
 12. The method of claim 1 wherein the molar ratio of saidfirst gas to said second gas is 1:100 to 100:1.
 13. The method of claim1 wherein the stabilized microbubble is used for diagnostic imaging. 14.A composition comprising stabilized microbubbles for in vivo use,comprising: a first gas, a second gas comprising a perfluorocarbon, amembrane forming in material, and a liquid, wherein said first gas andsaid second gas are present in a molar ratio of about 1:100 to about1,000:1, and wherein said second gas is a gas at 37 degrees C. and 760Hg with the proviso that said first gas and said second gas are notwater vapor; and said first gas and said second gas are surrounded withsaid membrane forming material to form stabilized microbubbles in saidliquid.
 15. The composition of claim 14 wherein the membrane formingmaterial comprises at least one surfactant.
 16. The composition of claim14 wherein the second gas is selected from the group consisting ofperfluoropropane, perfluorobutane, perfluorocyclobutane,perfluoromethylcyclobutane, perfluoropentane, perfluorocyclopentane,perfluoromethylcyclopentane, perfluorodimethylcyclobutanes,perfluorohexane, perfluorocyclohexane, perfluoroheptane,perfluorocycloheptane, perfluoromethylcyclohexane,perfluorodimethylcyclopentane, perfluorotrimethylcyclobutane, andperfluorotriethylamine.
 17. The composition of claim 14 wherein thefirst gas is selected from the group consisting of nitrogen, oxygen,carbon dioxide, air and mixtures thereof.
 18. The composition of claim14 wherein the membrane forming material is selected from the groupconsisting of albumin, phospholipid and polysaccharides.
 19. Thecomposition of claim 14 wherein the second gas is perfluorohexane andthe first gas is nitrogen.
 20. The composition of claim 14 wherein thesecond gas is pcrfluoropropane and the first gas is nitrogen.